Project Summary Possession of the ?4 allele of apolipoprotein E (APOE) is a major risk factor for late onset Alzheimer's disease (AD), although the direct cause remains a source of debate. Recent data has shown that the APOE gene is upregulated in a novel microglial activation state found in AD and other neurodegenerative diseases termed disease associated microglia (DAM). Furthermore, APOE4 expression has been shown to induce a neurotoxic activation of microglia in the presence of various stimuli, including tauopathy-afflicted neurons, although the mechanism of this activation has yet to be elucidated. In order to discover the role and mechanism of APOE4- associated microglial activation and its resulting neurotoxicity of tauopathy-afflicted neurons, I have devised an innovative set of experiments that utilize both cutting-edge technology and novel resources. In Aim 1, single- cell RNA-sequencing will be performed on microglia purified from a novel mouse line in which human APOE3 and APOE4 mice are crossed with EC-Tau mice, which express a pro-aggregating mutant tau protein primarily in the entorhinal cortex (EC). In Aim 2, APOE4's effects on microglial activation will be investigated in the setting of human microglia, using both a newly created isogenic APOE human stem cell line and powerful bioinformatics analyses. And in Aim 3, a robust in vitro co-culture assay will be utilized in order to both uncover the mechanism responsible for APOE4-associated microglial activation and the resulting neurotoxicity and to identify novel therapeutic strategies for inhibiting these events. As both APOE4 and microglial activation significantly impact AD, discovery of the mechanistic link between these two important players and therapeutic strategies for modulating APOE4-associated microglial activation would represent a major breakthrough in the AD field and would greatly advance our goal of preventing or slowing the progression of AD, especially among APOE4 carriers.